Annexin A1 modulates macula densa function by inhibiting cyclooxygenase 2.

Annexin A1 (ANXA1) exerts anti-inflammatory effects through multiple mechanisms including inhibition of prostaglandin synthesis. Once secreted, ANXA1 can bind to G protein-coupled formyl peptide receptors (Fpr) and activate diverse cellular signaling pathways. ANXA1 is known to be expressed in cells of the juxtaglomerular apparatus, but its relation to the expression of cyclooxygenase 2 (COX-2) in thick ascending limb and macula densa cells has not been elucidated. We hypothesized that ANXA1 regulates the biosynthesis of COX-2. ANXA1 abundance in rat kidney macula densa was extensively colocalized with COX-2 (95%). Furosemide, an established stimulus for COX-2 induction, caused enhanced expression of both ANXA1 and COX-2 with maintained colocalization (99%). In ANXA1-deficient mice, COX-2-positive cells were more numerous than in control mice (+107%; normalized to glomerular number; P < 0.05) and renin expression was increased (+566%; normalized to glomerular number; P < 0.05). Cultured macula densa cells transfected with full-length rat ANXA1 revealed downregulation of COX-2 mRNA (-59%; P < 0.05). Similarly, treatment with dexamethasone suppressed COX-2 mRNA in the cells (-49%; P < 0.05), while inducing ANXA1 mRNA (+56%; P < 0.05) and ANXA1 protein secretion. Inhibition of the ANXA-1 receptor Fpr1 with cyclosporin H blunted the effect of dexamethasone on COX-2 expression. These data show that ANXA1 exerts an inhibitory effect on COX-2 expression in the macula densa. ANXA1 may be a novel intrinsic modulator of renal juxtaglomerular regulation by inhibition of PGE(2) synthesis.

A(1) adenosine receptor-mediated PKC and p42/p44 MAPK signaling in mouse coronary artery smooth muscle cells.

The A(1) adenosine receptor (A(1)AR) is coupled to G(i)/G(o) proteins, but the downstream signaling pathways in smooth muscle cells are unclear. This study was performed in coronary artery smooth muscle cells (CASMCs) isolated from the mouse heart [A(1)AR wild type (A(1)WT) and A(1)AR knockout (A(1)KO)] to delineate A(1)AR signaling through the PKC pathway. In A(1)WT cells, treatment with (2S)-N(6)-(2-endo-norbornyl)adenosine (ENBA; 10(-5)M) increased A(1)AR expression by 150%, which was inhibited significantly by the A(1)AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (10(-6)M), but not in A(1)KO CASMCs. PKC isoforms were identified by Western blot analysis in the cytosolic and membrane fractions of cell homogenates of CASMCs. In A(1)WT and A(1)KO cells, significant levels of basal PKC-alpha were detected in the cytosolic fraction. Treatment with the A(1)AR agonist ENBA (10(-5)M) translocated PKC-alpha from the cytosolic to membrane fraction significantly in A(1)WT but not A(1)KO cells. Phospholipase C isoforms (betaI, betaIII, and gamma(1)) were analyzed using specific antibodies where ENBA treatment led to the increased expression of PLC-betaIII in A(1)WT CASMCs while having no effect in A(1)KO CASMCs. In A(1)WT cells, ENBA increased PKC-alpha expression and p42/p44 MAPK (ERK1/2) phospohorylation by 135% and 145%, respectively. These effects of ENBA were blocked by Gö-6976 (PKC-alpha inhibitor) and PD-98059 (p42/p44 MAPK inhibitor). We conclude that A(1)AR stimulation by ENBA activates the PKC-alpha signaling pathway, leading to p42/p44 MAPK phosphorylation in CASMCs.

AP214, an analogue of alpha-melanocyte-stimulating hormone, ameliorates sepsis-induced acute kidney injury and mortality.

Sepsis remains a serious problem in critically ill patients with the mortality increasing to over half when there is attendant acute kidney injury. alpha-Melanocyte-stimulating hormone is a potent anti-inflammatory cytokine that inhibits many forms of inflammation including that with acute kidney injury. We tested whether a new alpha-melanocyte-stimulating hormone analogue (AP214), which has increased binding affinity to melanocortin receptors, improves sepsis-induced kidney injury and mortality using a cecal ligation and puncture mouse model. In the lethal cecal ligation-puncture model of sepsis, severe hypotension and bradycardia resulted and AP214 attenuated acute kidney injury of the lethal model with a bell-shaped dose-response curve. An optimum AP214 dose reduced acute kidney injury even when it was administered 6 h after surgery and it significantly improved blood pressure and heart rate. AP214 reduced serum TNF-alpha and IL-10 levels with a bell-shaped dose-response curve. Additionally; NF-kappaB activation in the kidney and spleen, and splenocyte apoptosis were decreased by the treatment. AP214 significantly improved survival in both lethal and sublethal models. We have shown that AP214 improves hemodynamic failure, acute kidney injury, mortality and splenocyte apoptosis attenuating pro- and anti-inflammatory actions due to sepsis.

Axial and cellular heterogeneity in electrolyte transport pathways along the thick ascending limb.

The thick ascending limb (TAL) extends from the border of the inner medulla to the renal cortex, thus ascending through regions with wide differences in tissue solute and electrolyte concentrations. Structural and functional differences between TAL cells in the medulla (mTAL) and the cortex (cTAL) would therefore be useful to adapt TAL transport function to a changing external fluid composition. While mechanisms common to all TAL cells play a central role in the reclamation of about 25% of the NaCl filtered by the kidney, morphological features, Na+ / K+ -ATPase activity, NKCC2 splicing and phosphorylation do vary between segments and cells. The TAL contributes to K+ homeostasis and TAL cells with high or low basolateral K+ conductances have been identified which may be involved in K+ reabsorption and secretion respectively. Although transport rates for HCO3- do not differ between mTAL and cTAL, divergent axial and cellular expression of H+ transport proteins in TAL have been documented. The reabsorption of the divalent cations Ca2+ and Mg2+ is highest in cTAL and paralleled by differences in divalent cation permeability and the expression of select claudins. Morphologically, two cell types with different cell surface phenotypes have been described that still need to be linked to specific functional characteristics. The unique external environment and its change along the longitudinal axis require an axial functional heterogeneity for the TAL to optimally participate in conserving electrolyte homeostasis. Despite substantial progress in understanding TAL function, there are still considerable knowledge gaps that are just beginning to become bridged.

Interference with feedback control of glomerular filtration rate by furosemide, triflocin, and cyanide.

Microperfusion experiments have shown that increases in flow rate of tubule fluid through the loop of Henle are followed by reductions in single nephron glomerular filtration rate (SNGFR) and stop-flow pressure (SFP) measured in the proximal tubule of the same nephron. Because changes in luminal sodium concentration are not consistently related to changes in SNGFR and SFP, we explored the possibility that a transport step at a flow-dependent distal-sensing site might be involved in feedback control of SNGFR. Because the macula densa cells of the distal tubule are adjacent to the glomerular vessels of the same nephrons, they could be the distal-sensing mechanism. We perfused superficial loops of Henle from late proximal to early distal segments in three groups of rats while measuring SFP in the proximal tubule of the same nephron, SNGFR in the proximal tubule of the same nephron, or flow rates of fluid, Na, K, and Cl emerging from the perfused loops. Perfusion solutions used were 0.15 NaCl, Ringer or Ringer with one of several inhibitors of electrolyte transport. Perfusion rates were 10 or 40 nl/min (also, zero during measurements of SFP and SNGFR). With Ringer alone the loop-flow rate increased from 10 to 40 nl/min, caused a decrease in SFP from 37.6 to 32.1 mm Hg, and a decrease in SNGFR from 29.9 to 18.7 nl/min. Concentrations of Na, K, and Cl in early distal fluid and absorption of Na and Cl along the loop segment were also increased when loop perfusion rate was increased. Decreasing the perfusion rate to zero had little effect on SFP or SNGFR. The SFP response to increased flow rate did not occur when the perfusion solution contained furosemide (10(-4) M). No reduction of the SFP response was seen with other diuretics tested (amiloride, acetazolamide, ethacrynic acid, mercaptomerin) or with 0.15 M NaCl alone. The SNGFR response to increased perfusion rate was reduced by furosemide, triflocin, and cyanide but not by amiloride. Na and Cl absorption by the perfused segment were inhibited by furosemide, triflocin, cyanide, and amiloride. Amiloride and acetazolamide, probably do not act in the ascending limb. Ethacrynic acid and mercaptomerin are known to be ineffective in rat nephrons. Thus, agents that could have inhibited NaCl absorption by macula densa cells interfered with the feedback mechanism.

Tubuloglomerular feedback. Nonlinear relation between glomerular hydrostatic pressure and loop of henle perfusion rate.

The present experiments were performed to quantify the effect of changes in distal tubular sodium delivery on glomerular flow dynamics both below and above the normal physiologic range. Glomerular capillary pressure as derived from the tubular stop flow pressure was assessed while the loop of Henle of the same nephron was perfused with varying flow rates. During Ringer perfusion no change of glomerular capillary pressure was observed when flow was increased from 0 to 13 nl/min. Further increasing flow to 27 nl/min was associated with a reduction of glomerular hydrostatic pressure by an average of 7.0+/-4.4 cm H(2)O (+/-SD). During perfusion at a rate of 43 nl/min glomerular pressure was decreased by a mean of 10.5+/-4.0 cm H(2)O. Changing the flow rate in small steps revealed that a significant reduction of capillary pressure was found when increasing the flow rate from 13 to 21 nl/min and that the maximum response was reached at 32 nl/min. No effect of perfusion rate changes on glomerular capillary pressure was observed when 300 mM mannitol was used as perfusion fluid. These results imply that a nonlinear relationship exists between end-proximal flow rate and glomerular capillary pressure. It is suggested that during deviations of distal sodium delivery into a positive direction filtration rate is intrarenally regulated probably by prevalence of afferent arteriolar constriction. During reductions of distal sodium load intrarenal regulation is either abolished or it involves proportionate resistance changes of both afferent and efferent arterioles.

Effect of adenosine1-receptor blockade on renin release from rabbit isolated perfused juxtaglomerular apparatus.

Adenosine has been proposed to act within the juxtaglomerular apparatus (JGA) as a mediator of the inhibition of renin secretion produced by a high NaCl concentration at the macula densa. To test this hypothesis, we studied the effects of the adenosine1 (A1)-receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (CPX) on renin release from single isolated rabbit JGAs with macula densa perfused. The A1-receptor agonist, N6-cyclohexyladenosine (CHA), applied in the bathing solution at 10(-7) M, was found to inhibit renin secretion, an effect that was completely blocked by adding CPX (10(-5) M) to the bath. Applied to the lumen, 10(-5) M CPX produced a modest stimulation of renin secretion rates suppressed by a high NaCl concentration at the macula densa (P less than 0.05). The effect of changing luminal NaCl concentration on renin secretion rate was examined in the presence of CPX (10(-7) and 10(-5) M) in the bathing solution and in vehicle control experiments. The control response to increasing luminal NaCl concentration was a marked suppression of renin secretion, that was maintained as long as luminal NaCl concentration was high and was promptly reversible when concentration was lowered. CPX did not alter renin release when luminal NaCl was low, but diminished the reduction caused by high NaCl (P less than 0.01). It is concluded that A1-receptors are located within the JGA, and that A1-receptor activation inhibits renin release. A high NaCl concentration at the macula densa appears to influence A1-receptor activation, but a low NaCl concentration does not. The findings support participation of adenosine in macula densa control of renin secretion.

Direct vasoconstriction as a possible cause for amphotericin B-induced nephrotoxicity in rats.

In anesthetized rats we tested the hypothesis that amphotericin B (AmB) reduces glomerular filtration rate (GFR) by activating the tubuloglomerular feedback (TGF) mechanism. Infusion of 1 mg/kg AmB over 50 min was followed by a reduction in kidney GFR (from 0.47 +/- 0.03 to 0.39 +/- 0.02 ml/min per 100 g body wt during the second hour after infusion; P less than 0.05) and by an increase in urine flow and urinary chloride excretion. Single-nephron GFR (SNGFR) measured in proximal (TGF interrupted) or distal tubules (TGF intact) decreased to a similar degree from 33.4 +/- 1.8 and 30.6 +/- 1.2 nl/min in the control period to 19.7 +/- 1.9 and 21.2 +/- 1.6 nl/min during the second hour after AmB infusion (P less than 0.05). Distal chloride concentrations and TGF responses to changes in loop of Henle flow rate were not significantly altered by AmB. AmB at 10(-5) M reduced the diameter of isolated perfused afferent arterioles from rabbit kidneys. In isometrically contracting rings of rabbit aorta and renal artery in vitro AmB produced endothelium-independent constriction, with half-maximal contraction (EC50) being achieved by 1.8 x 10(-6) and 2.6 x 10(-6) M in intact vessels and 1.3 x 10(-6) and 1.7 x 10(-6) M in endothelium-denuded vessels respectively. Tension development did not occur in Ca-free media or in the presence of Ca channel blockers. Pretreatment with ouabain or Bay K 8644 potentiated the effect of AmB. The vasoconstrictive effect of AmB was counteracted by aminophylline and atrial natriuretic peptide. We conclude that the AmB-induced reduction in GFR is not caused by TGF activation and that AmB has a direct vasoconstrictor effect that is probably initiated by depolarization-induced opening of Ca channels. This effect may be an important component of the nephrotoxic actions of AmB.

Renin and renin mRNA in proximal tubules of the rat kidney.

The present study was undertaken to assess the presence of renin enzymatic activity and renin mRNA in proximal tubules of rat kidneys, and to determine the effect of converting enzyme inhibition (CEI) on proximal tubule renin gene expression. Proximal convoluted tubules (PCT), proximal straight tubules (PST), outer medullary collecting ducts (OMCD), and glomeruli (Gloms) were isolated by microdissection. Renin activity was measured in sonicated segments by radioimmunoassay. Renin mRNA levels were assessed using a quantitative PCR. Renin activity in PCT averaged 51 +/- 15 microGU/mm compared to 405 +/- 120 microGU/glomerulus. No measurable renin activity was found in PST and OMCD. Renin activity in both glomeruli and tubules had the same pH optimum, between 7.0 and 7.5. Renin mRNA was consistently detectable in cDNA prepared from PCT and PST, although its abundance per mm tubule was about 1/500th that found in one glomerulus. Renin mRNA was not detectable in OMCD. Tubular renin PCR product identity was confirmed by restriction digestion. CEI administration increased glomerular renin activity and renin mRNA, but not proximal tubular renin. The absence of a stimulatory effect of CEI on proximal tubule renin gene expression suggests the operation of different intracellular signals in control of renin synthesis in the proximal tubule than in the vascular compartment.

Intracellular ATP can regulate afferent arteriolar tone via ATP-sensitive K+ channels in the rabbit.

Studies were performed to assess whether ATP-sensitive K+ (KATP) channels on rabbit preglomerular vessels can influence afferent arteriolar (AA) tone. K+ channels with a slope conductance of 258 +/- 13 (n = 7) pS and pronounced voltage dependence were demonstrated in excised patches from vascular smooth muscle cells of microdissected preglomerular segments. Channel activity was markedly reduced by 1 mM ATP and in a dose-dependent fashion by glibenclamide (10(-9) M to 10(-6) M), a specific antagonist of KATP channels. 10(-5) M diazoxide, a K+ channel opener, activated these channels in the presence of ATP, and this effect was also blocked by glibenclamide. To determine the role of these KATP channels in the control of vascular tone, diazoxide was tested on isolated perfused AA. After preconstriction from a control diameter of 13.1 +/- 1.1 to 3.5 +/- 2.1 microns with phenylephrine (PE), addition of 10(-5) M diazoxide dilated vessels to 11.2 +/- 0.7 microns, which was not different from control. Further addition of 10(-5) M glibenclamide reconstricted the vessels to 5.8 +/- 1.5 microns (n = 5; P less than 0.03). In support of its specificity for KATP channels, glibenclamide did not reverse verapamil induced dilation in a separate series of experiments. To determine whether intracellular ATP levels can effect AA tone, studies were conducted to test the effect of the glycolytic inhibitor 2-deoxy-D-glucose. After preconstriction from 13.4 +/- 3.2 to 7.7 +/- 1.3 microns with PE, bath glucose was replaced with 6 mM 2-deoxy-D-glucose. Within 10 min, the arteriole dilated to a mean value of 11.8 +/- 1.4 microns (n = 6; NS compared to control). Subsequent addition of 10(-5) M glibenclamide significantly reconstricted the vessels to a diameter of 8.6 +/- 0.5 micron (P less than 0.04). These data demonstrate that KATP channels are present on the preglomerular vasculature and that changes in intracellular ATP can directly influence afferent arteriolar tone via these channels.

Reporter gene recombination in juxtaglomerular granular and collecting duct cells by human renin promoter-Cre recombinase transgene.

To assess the feasibility of using the renin promoter for expressing Cre recombinase in juxtaglomerular (JG) cells only, we generated five independent transgenic mouse lines (designated hRen-Cre) expressing Cre recombinase under control of a 12.2-kb human renin promoter. In the kidneys of adult mice Cre mRNA (RT-PCR) was found in the renal cortex, with Cre protein (immunohistochemistry) being localized in afferent arterioles and to a lower degree in interlobular arteries. Cre mRNA levels were regulated in a renin-typical fashion by changes in oral salt intake, water restriction, or isoproterenol infusion, indicating the presence of key regulatory elements within 12.2 kb of the 5'-flanking region of the human renin gene. hRen-Cre mice were interbred with both the ROSA26-EGFP and ROSA26-lacZ reporter strains to assess renin promoter activity from Cre-mediated excision of a floxed stop cassette and subsequent enhanced green fluorescent protein (EGFP) and beta-galactosidase (beta-gal) detection. In adult mice, beta-gal staining and EGFP were observed in afferent arterioles and interlobular arteries, overlapping with Cre protein expression. In addition, intense beta-gal staining was found in cortical and medullary collecting ducts where Cre expression was minimal. In embryonic kidneys, beta-gal staining was detected in the developing collecting duct system beginning at embryonic day 12, showing substantial activity of the human renin promoter in the branching ureteric bud. Our data indicate that besides its well-known activity in JG cells and renal vessels the human renin promoter is transiently active in the collecting duct system during kidney development, complicating the use of this approach for JG cell-specific excision of floxed targets.

Blood pressure, heart rate and tubuloglomerular feedback in A1AR-deficient mice with different genetic backgrounds.

AIM: Differences in genetic background between control mice and mice with targeted gene mutations have been recognized as a potential cause for phenotypic differences. In this study, we have used A1AR-deficient mice in a C57Bl/6 and SWR/J congenic background to assess the influence of background on the effect of A1AR-deficiency on cardiovascular and renal functional parameters. METHODS: In A1AR+/+ and A1AR-/- mice in C57Bl/6 and SWR/J congenic backgrounds, we assessed blood pressure and heart rate using radio-telemetry, plasma renin concentrations and tubuloglomerular feedback. RESULTS: We did not detect significant differences in arterial blood pressure (MAP) and heart rates (HR) between A1AR+/+ and A1AR-/- mice in either C57Bl/6, SWR/J or mixed backgrounds. MAP and HR were significantly higher in SWR/J than in C57Bl/6 mice. A high NaCl intake increased MAP in A1AR-/- mice on C57Bl/6 background while there was less or no salt sensitivity in the SWR/J background. No significant differences in plasma renin concentration were detected between A1AR-/- and A1AR+/+ mice in any of the strains. Tubuloglomerular feedback was found to be absent in A1AR-/- mice with SWR/J genetic background. CONCLUSIONS: While this study confirmed important differences between inbred mouse strains, we did not identify phenotypic modifications of A1AR-related effects on blood pressure, heart rate and plasma renin by differences in genetic background.

Regulation of endothelin production and secretion in cultured collecting duct cells by endogenous transforming growth factor-beta.

Confluent cultures of two renal collecting duct cell lines (M-1 and mIMCD-K2 cells derived from cortical and inner medullary collecting ducts, respectively) express endothelin1 (ET1), transforming growth factor-beta (TGF beta; both TGF beta 1 and TGF beta 2), and both types of the TGF beta receptor. Experiments were performed to test whether endogenous TGF beta may be a paracrine modulator of ET1 expression in these cells. Treatment of M-1 and mIMCD-K2 cells with TGF beta 2 antisense oligodeoxynucleotides (ODN) significantly reduced ET1 messenger RNA (mRNA) and ET secretion (as well as TGF beta 2 mRNA) in a concentration-dependent manner, whereas control ODN were without significant effects. To produce ET inhibition, antisense ODN had to be present in the basolateral medium, whereas its sole presence in the apical medium was without effect. In addition, a pan-specific TGF beta antibody caused a significant reduction of ET1 mRNA expression and ET1 secretion. M-1 cells were found to express high levels of the mRNA for plasminogen activator of both tissue and urokinase types. Addition of the nonspecific serine protease inhibitor aprotinin (50 micrograms/ml) to the medium for 24 h significantly reduced the secretion of ET1. These results suggest that secretion of endogenous TGF beta, at least in part activated by the plasminogen/plasmin system, participates in the regulation of ET1 synthesis and secretion by collecting duct cell lines.

Effects of furosemide and verapamil on the NaCl dependency of macula densa-mediated renin secretion.

The present studies in perfused specimens of the juxtaglomerular apparatus microdissected from rabbit kidneys were performed to quantitatively evaluate the relation between macula densa NaCl concentration and renin secretion and to study the effect of furosemide and verapamil on NaCl dependency of renin release. Renin secretion was found to decrease exponentially when macula densa NaCl concentration was increased from 26/7 mmol/L (Na/Cl) to 46/27, 66/47, and 86/67 mmol/L. Increasing Na/Cl concentrations from 86/67 to 106/87 mmol/L had no further effect on renin secretion. [Cl]1/2, the chloride concentration producing the half-maximal effect, was 30 mmol/L. Addition of 50 mumol/L furosemide to the luminal fluid caused renin secretion to become essentially independent of macula densa NaCl concentration. This effect was due to both an increase of renin secretion at high NaCl concentrations and a decrease of renin release at low NaCl concentrations. Verapamil added to the superfusate at a concentration of 1 mumol/L also abolished NaCl dependency of renin secretion; most of this effect was due to an increase of renin release at high luminal NaCl. These results suggest that Na-2Cl-K cotransport and calcium flux through voltage-gated channels are two mechanisms required for the expression of NaCl-dependent renin release. Identification of the cellular localizations of these two critical membrane proteins in the renin control pathway requires further study.

Time course of stimulation of renal renin messenger RNA by furosemide.

Renin secretion responds rapidly to a variety of stimuli; however, reported changes in renal renin messenger RNA (mRNA) levels in vivo have been observed only after prolonged stimulation. Studies were designed to test whether rapid changes in renin mRNA levels can be produced in vivo. In the first series, Sprague-Dawley rats received furosemide (10 mg/kg) intraperitoneally and a low sodium diet (0.05% sodium); renin secretion was significantly stimulated at 8 or 16 hours after treatment, but renin mRNA levels did not change. In a second series, rats were pretreated with deoxycorticosterone acetate (200 mg/kg) and saline drinking water for 3 days and then killed 0, 2, 4, 8, or 48 hours after furosemide administration. The renin mRNA level was unchanged at 2 hours but was stimulated twofold at 4 and 8 hours and threefold at 48 hours. In additional animals, the response of renin mRNA 4 hours after furosemide was found not to be potentiated by the converting enzyme inhibitor quinapril (5 mg/kg). The results demonstrate that with acute stimulation, renin mRNA levels lag 2-4 hours behind the change in plasma renin levels.

SA gene expression in the proximal tubule of normotensive and hypertensive rats.

Previous studies have shown that the SA gene is expressed at higher levels in the kidney of genetically hypertensive rats than in control strains and that in hybrid crosses of genetically hypertensive rats and normotensive controls, markers in or close to the SA gene cosegregate with blood pressure. The present studies examine the localization of the SA gene product in the kidney by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). cDNA was prepared from microdissected nephron segments from Sprague-Dawley (SD) rats, spontaneously hypertensive rats (SHRs), and Wistar-Kyoto (WKY) rats, and RT-PCR was performed using specific primers. In all three strains, SA gene mRNA was found to be abundantly expressed in proximal tubules. SA PCR product was occasionally detected at approximately 100-fold lower abundance in glomeruli, while no signal was obtained from the collecting duct, thick ascending limb of the loop of Henle, or arcuate artery. Within the proximal tubule of normotensive rats, distribution of SA mRNA was found to be strain dependent: in SD rats it was expressed at high levels in the proximal convoluted tubule, whereas in WKY rats it was restricted to the proximal straight tubule. In SHRs, SA PCR product was detected along the entire proximal tubule. Induction of hypertension by renal artery clamping (two-kidney, one-clamp Goldblatt model) did not alter the pattern of expression observed in the SD rat. These results indicate that an extension of SA gene expression to the full length of the proximal tubule accompanies spontaneous hypertension and that in nonhypertensive animals the pattern of gene product expression is more restricted but shows substantial strain variability.

Effect of dopamine antagonists on the urine flow of rats infused with hypotonic saline.

1. The probable involvement of dopamine in the regulation of water excretion was investigated by administering dopamine antagonists intravenously to barbiturate--anaesthetized rats undergoing a water diuresis induced by the infusion of 0.83% glucose with 0.3% NaCl at the rate of 9 ml h-1. 2. Administration of 100 micrograms of the D1-/D2-dopamine antagonist, haloperidol, reduced the enhanced urine flow of rats infused with the hypotonic solution by 69% (from 75.4 +/- 13.0 to 23.6 +/- 6.0 microliter min-1, P less than 0.01). Similarly, the D1-receptor antagonist, SCH 23390, reduced urine flow by 58% (from 77.5 +/- 9.2 to 32.7 +/- 7.2 microliters min-1, P less than 0.01) and the D2-receptor antagonist, sulpiride, by 47% (from 66.2 +/- 8.6 to 35.1 +/- 6.8 microliter min-1, P less than 0.05). 3. The injection of SCH 23390 increased the urine osmolality from 189.6 +/- 27.5 to 479.8 +/- 45.8 mosm kg-1 (P less than 0.05). There was no significant change in sodium and potassium excretion in any of the experiments. Blood pressure (BP) decreased after haloperidol and SCH 23390 injection from control values of 121.7 +/- 1.7 and 116.5 +/- 7.4 to 113.3 +/- 3.3 and 106.0 +/- 8.8 mmHg respectively (P less than 0.05). 4. To study whether the influence of dopamine antagonists on urine flow during water diuresis depends on antidiuretic hormone (ADH), we administered 0.6 micrograms d(CH2)5-D-Phe-Ile-AVP (an ADH antagonist) shortly after the injection of 100 micrograms SCH 23390. The preferential V2 ADH-antagonist abolished the antidiuretic effect of SCH 23390 but did not affect its blood pressure reducing effect (from 118.6 +/- 5.6 to 103.2 +/- 4.6 mmHg, P <0.01). 5. These results suggest that dopamine antagonists blunted the hypotonic saline-induced diuresis by favouring ADH release through an interference with an inhibitory dopaminergic pathway.

Cellular mechanisms within the juxtaglomerular apparatus.

The tubular-vascular connection via the juxtaglomerular apparatus appears to serve two functions, local control of renal vascular resistance and regulation of renin secretion. A fall in single nephron glomerular filtration rate (SNGFR) and an increase in resistance are produced by an increase in NaCl concentration at the macular densa. This change also results in inhibition of secretion of renin. The macula densa has a unique location near the terminal end of the thick ascending limb, where NaCl concentration is highly flow dependent. The cellular mechanisms by which changes in tubular fluid NaCl produce vasoconstriction and inhibition of renin secretion are unknown, but the anatomy of the juxtaglomerular apparatus strongly suggests that such responses may be mediated by the extraglomerular mesangial cells located in the polar cushion underlying the macula densa. Recent evidence suggests that interstitial chloride concentration in this compartment may be quite variable, and that increases in external chloride may enhance the activation of the mesangial cell.

Concentration-dependent sodium chloride transport as the signal in feedback control of glomerular filtration rate.

There is good evidence that the initial step leading to a feedback response is the concentration-dependent active transport of sodium chloride across the macula densa cells. This may lead to changes in sodium chloride concentration or tonicity in the small and relatively unstirred compartment of the juxtaglomerular interstitium or to changes in the concentration of transport-related compounds or metabolites. We assume that the Goormaghtigh cells act as receptor cells that transform such compositional changes into a signal propagating to the glomerular vascular elements.

Effect of angiotensin and other pressor agents on tubuloglomerular feedback responses.

Experiments were performed in anesthetized rats to examine the effect of an intravenous infusion of norepinephrine or vasopressin on the tubuloglomerular feedback (TGF) response of stop flow pressure (PSF). During infusion of norepinephrine at an average rate of 107.5 ng/kg min, mean femoral arterial pressure (MAP) increased from 102.1 +/- 3.55 to 113.7 +/- 3.44 mm Hg and PSF-max increased from 7.45 +/- 1.13 to 9.95 +/- 1.19 mm Hg. When MAP was returned to control by a suprarenal aortic clamp PSF-max was 5.64 +/- 1.09 mm Hg (NS vs. control). Similarly, at an infusion rate of 226.5 ng/kg min PSF-max was not significantly different from control (6.79 +/- 1.61 mm Hg). V1/2, the half-maximum flow rate, was not altered by norepinephrine whether MAP increased or was kept constant. Infusion of vasopressin at the pressor dose of 13.0 mU/kg min increased MAP by about 25 mm Hg and raised PSF-max from 6.56 +/- 0.84 to 14.45 +/- 1.54 mm Hg. However, when MAP was returned to normal PSF-max was 5.41 +/- 0.75 mm Hg (NS). Our data show that in contrast to angiotensin II, norepinephrine and vasopressin do not augment TGF responses when a rise in MAP is prevented. Angiotensin II appears to play a specific role in altering the sensitivity of the TGF mechanism.